Journal: Human Molecular Genetics

Article Title: PD-linked CHCHD2 mutations impair CHCHD10 and MICOS complex leading to mitochondria dysfunction

doi: 10.1093/hmg/ddy413

Figure Lengend Snippet: PD-linked CHCHD2 mutants showed reduced binding to CHCHD10. ( A ) Co-immunoprecipitation of endogenous CHCHD2 by antibody against CHCHD2 in SK-N-SH cells. WCL: whole cell lysate, 5% total protein used in co-IP experiment. ( B ) Co-immunoprecipitation of endogenous CHCHD10 by antibody against CHCHD10 in SK-N-SH cells. WCL: whole cell lysate, 5% total protein used in co-IP experiment. ( C ) Co-immunoprecipitation of endogenous CHCHD2 by antibody against CHCHD2 in human brain tissue lysates. ( D ) Co-immunoprecipitation of CHCHD2 by a CHCHD2 antibody against middle region of CHCHD2 (labeled as CHCHD2-TF) in SK-N-SH cells transfected with non-tagged CHCHD2 WT/T61I/R145Q/Q126X. WCL: whole cell lysate, 5% total protein used in co-IP experiment. Lower arrow pointed to CHCHD2 Q126X and higher arrow pointed to full length CHCHD2. ( E ) Co-immunoprecipitation of CHCHD2 by a CHCHD2 antibody against middle region of CHCHD2 (labeled as CHCHD2-TF) in hESCs of H9 and isogenic lines harboring homozygous R145Q (−/−).WCL: whole cell lysate, 5% total protein used in co-IP experiment. Arrow pointed to CHCHD10. Representative results from R10 and R17 (R145Q−/−) were shown. ( F ) Quantification of protein abundance of CHCHD10 normalized with CHCHD2 on the co-immunoprecipitation complex from isogenic hESC lysates.

Article Snippet: Human brain tissue lysates were from Novus Centennial, CO. Elamipretide was from MedChemExpress Monmouth Junction, NJ.

Techniques: Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Labeling, Transfection, Quantitative Proteomics

Journal: Journal of Neuroinflammation

Article Title: A selective inhibitor of the NLRP3 inflammasome as a potential therapeutic approach for neuroprotection in a transgenic mouse model of Huntington’s disease

doi: 10.1186/s12974-022-02419-9

Figure Lengend Snippet: MCC950 markedly reduces cytotoxicity in striatal progenitor cells and BV2 microglial cells. A , B BV2 microglia were incubated for 4 h with LPS (1 μg/mL) followed by incubation with MCC950 (1 μM) for 2 h. The cells were then incubated with ATP (1 mM, 24 h). Total lysates of BV2 microglial cells were assessed by Western blot analysis to determine the levels of the NLRP3 and actin proteins. The molecular mass is indicated in kilodaltons. C , D BV2 microglia were incubated for 4 h with LPS (1 μg/mL) followed by incubation with MCC950 (1 μM) for 2 h. The cells were then incubated with ATP (1 mM for 24 h). Cell survival ( C ) and IL-1β expression levels ( D ) were measured using the CCK-8 assay and ELISA, respectively. The values of the indicated cells were normalized to those of untreated BV2 cells. * P < 0.05 compared to LPS/ATP treated cells ( n = 3). E , F ST Hdh Q109 cells were incubated for 24 h with MCC950 (1 μM). Total lysates of ST Hdh Q7 and ST Hdh Q109 cells were assessed using Western blot analysis. G ST Hdh Q7 and ST Hdh Q109 cells were incubated for 24 h with MCC950 (1 μM). Cell death was quantified using the CCK-8 assay; the values of the indicated cells were normalized to those of untreated ST Hdh Q7 cells. The data are presented as the mean ± SEM from three independent experiments. * P < 0.05, ST Hdh Q7 vs. ST Hdh Q109 cells; # P < 0.05 vs. untreated ST Hdh Q109 cells. H BV2 cells were incubated with LPS (1 µg/mL for 4 h) with or without MCC950 for 2 h before stimulation with ATP (1 mM for 24 h). The BV2 medium was then collected and used to culture the ST Hdh Q109 cells for an additional 24 h. ST Hdh Q7 and ST Hdh Q109 cell viability was determined by CCK-8 assay. The data are presented as the mean ± SEM from three independent experiments. * P < 0.05, ST Hdh Q7 vs. ST Hdh Q109 cells; # P < 0.05 vs. untreated ST Hdh Q109 cells. I BV2 cells were treated with or without MCC950 (1 μM) and 3-NP (5, 10, and 20 mM) for 24 h. The cell viability was determined using the CCK-8 assay. Data are presented as the mean ± SEM from three independent experiments. * P < 0.05 compared with controls ( n = 3)

Article Snippet: BV2 cells (mouse, C57BL/6; brain, microglial cells) were purchased from the American Type Culture Collection (Rockville, MD).

Techniques: Incubation, Western Blot, Expressing, CCK-8 Assay, Enzyme-linked Immunosorbent Assay